Method of detecting gas-producing microbial colonies

ABSTRACT

A first method comprises using an imaging device to produce a plurality of images of a culture device, analyzing a first image to identify a microorganism colony at a first location, analyzing a second image to identify a gas bubble at a second location, and determining whether the first location is proximate the second location. A second method comprises analyzing an image of a culture device to detect gas bubbles and classifying the gas bubbles according to a size parameter associated with each of the gas bubbles. A third method comprises analyzing a first area of an image of a culture device to detect a first number of gas bubbles, analyzing a second area of the image to detect a second number of gas bubbles, and comparing the first number of gas bubbles to the second number of gas bubbles.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/652,379, filed Jun. 15, 2015, which is a national stage filing under35 U.S.C. 371 of International Patent Application No. PCT/US2013/074887,filed Dec. 13, 2013, which claims priority to U.S. Provisional PatentApplication No. 61/739,789, filed Dec. 20, 2012, the disclosures ofwhich are incorporated by reference in their entirety herein.

BACKGROUND

Biological safety is a paramount concern in modern society. Testing forbiological contamination in foods or other materials has become animportant and often mandatory requirement for developers anddistributors of food products. Biological testing is also used toidentify bacteria or other agents in laboratory samples such as bloodsamples taken from medical patients, laboratory samples developed forexperimental purposes, and other types of biological samples. Varioustechniques and devices can be utilized to improve biological testing andto streamline and standardize the biological testing process.

A wide variety of culture devices have been developed. As one example,culture devices have been developed by 3M Company (hereafter “3M”) ofSt. Paul, Minn. In particular, culture devices are sold by 3M under thetrade name PETRIFILM plates. Culture devices can be utilized tofacilitate the rapid growth and detection of microorganisms commonlyassociated with food contamination, including, for example, aerobicbacteria, E. coli, coliform, enterobacteria, yeast, mold, Staphylococcusaureus, Listeria, Campylobacter, and the like. The use of PETRIFILMplates, or other growth media, can simplify bacterial testing of foodsamples.

Culture devices can be used to enumerate or identify the presence ofbacteria so that corrective measures can be performed (in the case offood testing) or proper diagnosis can be made (in the case of medicaluse). In other applications, culture devices may be used to rapidly growmicroorganisms in laboratory samples, e.g., for experimental purposes.

Biological scanning units refer to devices used to scan and/or countmicrobial colonies. For example, a food sample or laboratory sample canbe placed on a culture device, and then the plate can be inserted intoan incubation chamber. After incubation, the culture device can beplaced into the biological scanning unit for automated detection andenumeration of bacterial growth. In this manner, biological scanningunits automate the detection and enumeration of microbial colonies in aculture device, and thereby improve the biological testing process byreducing human error.

SUMMARY

In general, the present disclosure is directed to a technique fordistinguishing objects in a scanned image. In particular, the techniqueis used to identify a gas-producing microorganism colony that is presentin an image of a culture device, the culture device comprising a culturemedium disposed without a headspace between two substrates. In addition,the technique further may be used to count the number of gas-producingmicrobial colonies in the scanned image of the culture device. To countthe colonies, a culture device containing the culture medium is insertedinto a scanning unit. Upon insertion of the culture device, the scanningunit generates an image of the culture device. Then, the number ofgas-producing microorganism colonies can be counted or otherwisedetermined using image processing and analysis routines performed eitherwithin the scanning unit or by an external computing device, such as adesktop computer, workstation or the like. In accordance with theinvention, a method of distinguishing a gas-producing colony isdescribed. The method can be used to improve the accuracy over existingmethods of automated counts of microorganism colonies in a scannedimage.

In one aspect, the present disclosure provides a method. The method cancomprise using an imaging device to produce a first image of a thin filmculture device, the culture device having a front side having atransparent film cover sheet and a back side having a translucentsubstrate. The first image is produced while providing illumination tothe front side of the device. The culture device comprises an indicatorcompound that is converted by a microorganism to a first product that isobservable by a first color. The culture device comprises a nutrientthat can be converted to by a first type of microorganism to a gas. Themethod further can comprise using the imaging device to produce a secondimage of the thin film culture device, wherein the second image isproduced while providing illumination to the back side of the device;analyzing the first image to identify a microorganism colony at a firstlocation in the culture device; analyzing the second image to identify afirst gas bubble at a second location in the culture device; anddetermining whether the first location is within a predetermineddistance from the second location.

In any embodiment, the first image can be produced while illuminatingthe device with a first ratio of front-side illumination to back-sideillumination, wherein the second image can be produced whileilluminating the device with a second ratio of front-side illuminationto back-side illumination that is lower than the first ratio. In any ofthe above embodiments, the first gas bubble can comprise a firstperimeter, wherein analyzing the second image to identify a gas bubblecan comprise identifying a dark annulus associated with the firstperimeter. In any of the above embodiments, analyzing the second imageto identify a first gas bubble can comprise calculating a size parameterof the first gas bubble. In any of the above embodiments, the methodfurther can comprise comparing the size parameter of the first gasbubble to the size parameter of a second gas bubble. In any of the aboveembodiments, the method further can comprise using the first image tocount a number of microorganism colonies in the culture device. In anyof the above embodiments, the method further can comprise using thefirst and second images to count a number of microorganism colonies thatdon't convert the nutrient to a gas.

In another aspect, the present disclosure provides a computer readablemedium comprising computer readable instructions. The computer readableinstructions, when executed by a processor, can cause an image-analyzingsystem comprising the processor to analyze a first image of a thin filmculture device, the culture device having a front side and a back sideopposite the front side. The first image is produced while providingillumination to the front side of the device. Analyzing the first imagecomprises identifying a microorganism colony at a first location in theculture device. The computer readable instructions, when executed by aprocessor, further can cause the processor to analyze a second image ofthe thin film culture device, wherein the second image is produced whileproviding illumination to the back side of the device. Analyzing thesecond image comprises identifying a gas bubble at a second location inthe culture device. The computer readable instructions, when executed bya processor, further can cause the processor to determine whether thefirst location is within a predetermined distance from the secondlocation.

In any of the above embodiments of the computer readable medium,analyzing the second image to identify a second location of a gas bubblecan comprise identifying a dark annulus surrounding the gas bubble. Inany of the above embodiments, the computer readable medium furthercomprise instructions that, when executed in the processor, can causethe system to use the first image to count a number of microorganismcolonies in the culture device. In any of the above embodiments, thecomputer readable medium further comprise instructions that, whenexecuted in the processor, can cause the system to use the first andsecond images to count a number of gas-producing microorganism coloniesin the culture device.

In yet another aspect, the present disclosure provides a method. Themethod can comprise analyzing an image of the growth area of a thin filmculture device to detect gas bubbles and classifying a plurality of thegas bubbles, wherein classifying the plurality of gas bubbles comprisesassigning each gas bubble to a first group or a second group accordingto a size parameter associated with each of the plurality of gasbubbles. In any embodiment, classifying the gas bubbles into a firstgroup and a second group can comprise classifying a first subset of gasbubbles into a first suspect abiogenic bubble group and the methodfurther can comprise assigning a size parameter value upper limit forthe first suspect abiogenic bubble group. In any embodiment, classifyingthe gas bubbles into a first group and a second group can compriseclassifying a second subset of gas bubbles into a suspect biogenicbubble group and the method further can comprise assigning a sizeparameter value lower limit for the suspect biogenic bubble group.

In yet another aspect, the present disclosure provides a computerreadable medium. The computer readable medium comprises computerreadable instructions that, when executed by a processor can cause animage-analyzing system comprising the processor to analyze an image ofthe growth area of a thin film culture device to detect gas bubbles andto classify a plurality of the gas bubbles, wherein classifying theplurality of gas bubbles comprises assigning each gas bubble to a firstgroup or a second group according to a size parameter associated witheach of the plurality of gas bubbles. In any embodiment, classifying thegas bubbles into a first group and a second group can compriseclassifying a first subset of gas bubbles into a first suspect abiogenicbubble group and the computer readable instructions further can causethe processor to assign a size parameter value upper limit for the firstsuspect abiogenic bubble group. In any embodiment, classifying the gasbubbles into a first group and a second group can comprise classifying asecond subset of gas bubbles into a suspect biogenic bubble group andthe computer readable instructions further can cause the processor toassign a size parameter value lower limit for the suspect biogenicbubble group.

In yet another aspect, the present disclosure provides a method. Themethod can comprise analyzing a first area of an image of a growth areaof a thin film culture device to detect a first number of gas bubbles inthe first area, analyzing a second area of the image to detect a secondnumber of gas bubbles in the second area, and comparing the first numberof gas bubbles to the second number of gas bubbles. In any embodiment,the method further can comprise analyzing a third area of the image todetect a third number of gas bubbles in the third area and comparing thefirst number of gas bubbles or second number of gas bubbles to the thirdnumber of gas bubbles. In any embodiment, the method further cancomprise analyzing the image to detect gas bubbles in the growth area ofthe culture device and classifying a plurality of the gas bubbles,wherein classifying the plurality of gas bubbles comprises assigningeach gas bubble to a first group or a second group according to a sizeparameter associated with each of the plurality of gas bubbles. In someembodiment, the method further can comprise determining whether a gasbubble in any of the first, second, or third areas is assigned to thefirst group or the second group.

In yet another aspect, the present disclosure provides a computerreadable medium. The computer readable medium comprises computerreadable instructions that, when executed by a processor can cause animage-analyzing system comprising the processor to analyze a first areaof an image of a growth area of a thin film culture device to detect afirst number of gas bubbles in the first area, analyze a second area ofthe image to detect a second number of gas bubbles in the second area,and compare the first number of gas bubbles to the second number of gasbubbles. In any embodiment, the computer readable instructions furthercan cause the processor to analyze a third area of the image to detect athird number of gas bubbles in the third area and to compare the firstnumber of gas bubbles or second number of gas bubbles to the thirdnumber of gas bubbles. In any embodiment, the computer readableinstructions further can cause the processor to analyze the image todetect gas bubbles in the growth area of the culture device and toclassify a plurality of the gas bubbles, wherein classifying theplurality of gas bubbles comprises assigning each gas bubble to a firstgroup or a second group according to a size parameter associated witheach of the plurality of gas bubbles. In any embodiment, the computerreadable instructions further can cause the processor to determinewhether a gas bubble in any of the first, second, or third areas isassigned to the first group or the second group.

Various aspects of the invention may provide a number of advantages. Forexample, the invention may improve the accuracy of automated counts ofmicrobial colonies on a culture device. In particular, the counting ruledescribed herein may address problems that commonly occur, and which canotherwise undermine the accuracy of automated counting of microbialcolonies in culture device.

Additional details of these and other embodiments are set forth in theaccompanying drawings and the description below. Other features, objectsand advantages will become apparent from the description and drawings,and from the claims.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a perspective view of an exemplary system comprising ascanning device coupled to an external computer which performs imaginganalysis of the images generated by the scanning device.

FIG. 2 is a block diagram of a biological scanning system that maycorrespond to the system illustrated in FIG. 1.

FIG. 3 is a flow diagram illustrating a process of automated analysis ofa microbial culture device.

FIG. 4 is a block diagram of one embodiment of a method of analyzing amicrobial culture device according to the present disclosure.

FIG. 5 is a flow diagram of a counting rule to distinguish gas-producingmicrobial colonies according to the present disclosure.

FIG. 6 is a black-and-white image of a portion of a thin film culturedevice, wherein the image was obtained while illuminating only the backside of the culture device.

FIG. 7 is a black and white image of the portion of the thin filmculture device of FIG. 6, wherein the image was obtained whileilluminating only the front side of the culture device.

FIG. 8 is a graph of the relative intensities of red, green, and bluecomponents, respectively, of the pixels in the line scan of FIG. 6.

FIG. 9 is a graph of the relative intensities of red, green, and bluecomponents, respectively, of the pixels in the line scan of FIG. 7.

FIG. 10A is a black-and-white image of a portion of a thin film culturedevice showing gas-producing and non-gas producing colonies ofmicroorganisms, wherein the image was obtained while illuminating onlythe back side of the culture device.

FIG. 10B is the black-and-white image of FIG. 10A showing a first maskedarea of the image.

FIG. 10C is the black-and-white image of FIG. 10A showing a secondmasked area of the image.

FIG. 10D is the black-and-white image of FIG. 10A showing a third maskedarea of the image.

DETAILED DESCRIPTION

Detection and counting of microorganisms is a universal problem in manydiverse fields. Microorganisms occur in almost all foods, in water, inair, and on numerous surfaces and substances with which humans come incontact. Such microorganisms are often harmful and therefore must bemeasured and controlled.

A widely used practice for detecting the presence of microorganisms in asubstance (e.g., food, water, environmental residue) is to place asample of the substance to be tested, suitably prepared, in a culturedevice, and to allow the microorganisms to grow into colonies. Whencultured in such a medium, colonies become visible to the eye and can becounted. Each visible colony corresponds to one original microorganism.A method of the present disclosure is performed using such culturedevices for growing and counting microbial colonies. The culture deviceincludes an aqueous nutrient medium distributed in a matrix (e.g., agelling agent such as agar, guar gum, or pectin, for example) tomaintain separation of individual colonies. Many culture devices furtherinclude indicator compounds as discussed herein. Suitable culturedevices for growing and detecting gas-producing microbial coloniesaccording to the present disclosure include culture devices thatcomprise a culture medium disposed between two substrates. That is, theculture medium is sandwiched between two substrates without a headspacebetween the culture medium and either of the two substrates.

Suitable culture devices for use in the method of the present disclosureinclude, for example, thin film culture devices sold by 3M Company underthe PETRIFILM trade name. PETRIFILM thin film culture devices aredisclosed in numerous publications including, for example, U.S. Pat.Nos. 5,364,766; 5,601,998; and 5,681,712; which are all incorporatedherein by reference in their entirety.

Many culture media, including typical agar culture media and culturemedia used in PETRIFILM plates, include indicator compounds to indicatethe presence of a microorganism. Indicator compounds include, forexample, pH indicators, chromogenic enzyme substrates, and redoxindicators. The indicator compounds, when converted directly orindirectly to a product, typically impart a color change to themicrobial colony and/or the culture medium surrounding the colony. Thecolor change often makes it easier to detect the presence of themicrobial colony in the culture medium (e.g., it improves the colorcontrast between the colony and the culture medium) and may the colorchange also may serve to differentiate a particular colony that reactswith a particular indicator compound from another microbial colony thatdoes not react with that indicator compound.

Many types of culture media for growing and differentiatingmicroorganisms include two or more indicator compounds. For example, theculture medium in a PETRIFILM E. coli Count Plate, when hydrated with anaqueous buffer and/or sample, contains a redox indicator(triphenyltetrazolium chloride, hereinafter “TTC”) and a chromogenicenzyme substrate (5-bromo-4-chloro-3-indolyl-β-D-glucuronide,hereinafter “X-gluc”). The TTC reacts with microbial cells to form areddish-colored formazan that stains the cell mass of any bacterialcolony that grows in the Gram-negative selective growth medium. Incontrast, the X-gluc reacts only with bacteria that, in addition tobeing able to grow in the selective growth medium, possessβ-D-glucuronidase enzyme activity (e.g., E. coli strains that possessβ-D-glucuronidase enzyme activity). Hydrolysis of X-gluc causes theformation of an indigo dye, which stains the cell mass of the colonyblue and forms a less intensely-blue halo surrounding the colony havingβ-D-glucuronidase enzyme activity.

It is contemplated that the method of the present disclosure can be usedto distinguish microbial colonies on the basis of their reaction withone or more of a plurality of indicator compounds and by theirrespective gas production or lack thereof. The microbial colony canreact with one or more indicator compounds to produce a colored orfluorescent product that indicates the presence of the microbes. Inaddition, the presence of a gas bubble associated with a microbialcolony indicates the colony belongs to a group of microorganisms capableof metabolizing a nutrient present in the culture device to a gaseousend product (e.g., carbon dioxide).

Thus, a culture device used in a method according to the presentdisclosure comprises an effective amount of a nutrient that can beconverted (e.g., by fermentation) to a gaseous end product (e.g., carbondioxide). The culture device also has a unique structure (i.e., ahydrated gel sandwiched between two substantially planar layers) suchthat a gas bubble produced by microbial activity is trapped in theculture device substantially displaces a portion of the hydrated gel,thereby forming an optically-detectable void space between the planarlayers. In any embodiment, the nutrient can be a carbohydrate such asglucose, sucrose, lactose, or a combination of any two or more of theforegoing carbohydrates, for example. A person having ordinary skill inthe art will recognize a variety of nutrients that are converted bymicrobial activity to a gaseous end product and that can be used in aculture medium to identify gas-producing colonies according to thepresent disclosure.

A culture medium used in a method of the present disclosure can beselected so that it favors the growth of one type of microorganism overother types of microorganisms. For example, the culture medium mayinclude selective ingredients (e.g., antibiotics, sodium chloride, bilesalts, dyes such as crystal violet, for example) that inhibit the growthof certain microorganisms (e.g., Gram-negative bacteria) and/or favorthe growth of other microorganisms. A person having ordinary skill inthe art will recognize a variety of selective agents that can be used tofacilitate the growth of certain microorganisms in a culture device usedaccording to a method of the present disclosure.

According to the method of the present disclosure, the sample isprepared, inoculated into the culture device, and incubated according toprocedures that are well known in the art. Sample preparation mayoptionally include dilution, enzymatic digestion, filtration, and/orsedimentation to reduce or remove nonmicrobial debris from the sampleprior to introducing the sample into (e.g., pour-plating) or onto (e.g.,surface-plating) the nutrient medium in the culture device.

After a sufficient incubation period at a temperature suitable for thegrowth of the microorganisms suspected of being present in the sample,microbial colonies can be detected and counted using an imaging systemto capture an image of microbial colonies in a culture device andapplying various image-analysis schemes. Examples of imaging systemsused to count and/or differentiate microbial colonies in a culturedevice can be found in International Publication No. WO 98/59314; andU.S. Pat. Nos. 7,298,885; 8,094,916; and 7,496,225; which areincorporated herein by reference in their entirety. Examples of imageanalysis schemes to detect and/or enumerate microbial colonies in aculture device can be found in U.S. Pat. Nos. 6,058,209 and 6,243,486,which are incorporated herein by reference in their entirety.

The present disclosure is directed to techniques for counting microbialcolonies in an image of a culture device (e.g., a thin film culturedevice). The techniques can be used to improve the accuracy of automatedcounts of microbial colonies in a culture device. The counting rulesdisclosed herein may be used individually, or may be used in combinationwith other counting rules such as, for example, the counting rulesdisclosed in International Patent Publication No. WO 2005/062744 and inU.S. Provisional Patent Application No. 61/739,786, filed Dec. 20, 2012and entitled “METHOD OF DIFFERENTIATING MICROBIAL COLONIES IN AN IMAGE”,which are both incorporated herein by reference in their entirety.

The counting rules disclosed herein are typically stored ascomputer-executable software instructions, and are executed by aprocessor in a biological scanning system or an image-analyzing system(e.g., a processor and image-analysis software, optionally coupled to ascanning device). Alternatively, the rules may be implemented inhardware such as an application specific integrated circuit (ASIC), afield programmable gate array (FPGA), or various hardware componentsknown in the art. The rules described herein may be appliedindividually, or in any combination with other counting rules dependingon the growth medium being scanned. In any case, by applying the rulesdescribed herein, the accuracy of automated counts of microbial colonieson a culture device (e.g., a thin film culture device) can be improved.

In any embodiment, a method of the present disclosure employs a systemfor detecting and counting microbial colonies in a culture device.Systems for detecting and counting microbial colonies in a culturedevice are described, for example, in International Patent PublicationNos. WO 96/18720, WO 96/18167, WO 2005/062744, which are allincorporated herein by reference in their entirety.

FIG. 1 shows a perspective view of one embodiment of a system 20 fordetecting and counting microbial colonies in a culture device. Thesystem 20 comprises a scanner 21 coupled to an external computer 22which performs imaging analysis of the images generated by the scanner.External computer 22 may include, for example, a microprocessorprogrammed for image analysis of a culture device 24. External computer22 may comprise a personal computer (PC), desktop computer, laptopcomputer, handheld computer, workstation, tablet personal computingdevice, mobile device or the like. For example, software programs can beloaded on external computer 22 to facilitate image analysis of images ofculture device 24 generated by scanner 21.

Scanner 21 is coupled to external computer 22 via interface 25.Interface 25, for example, may comprise a Universal Serial Bus (USB)interface, a Universal Serial Bus 2 (USB2) interface, an IEEE 1394 FireWire interface, a Small Computer System Interface (SCSI) interface, anAdvance Technology Attachment (ATA) interface, a serial ATA interface, aPeripheral Component Interconnect (PCI) interface, a conventional serialor parallel interface, wireless connection or the like.

The culture device 24 optionally may include indicia 29, such as a barcode or other type of identification marking used to identify culturedevice 24. RFID tags, two-dimensional optically detectable codes, or thelike, may also be used as indicia. In any case, indicia 29 may identifythe type of microorganism being grown and tested on the culture device24. Scanner 21 can be designed to draw the culture device 24 intoscanner 21 to a first location and generate an image of indicia 29, andthen draw the culture device 24 to a second location and generate animage of the growth area 27. In this manner, images of indicia 29 andgrowth area 27 of the culture device can be generated by system 20.Alternatively, a single image may capture both indicia 29 and the growtharea 27. In either case, the scanning of indicia 29 can facilitateidentification of the type of plate being used so that one or moredesirable counting rules can be applied in an automated fashion.

By way of example, the culture device 24 may comprise a thin filmculture device sold by 3M under the trade name PETRIFILM plates. Culturedevice 24 can be utilized to facilitate the rapid growth and detectionof microorganisms commonly associated with food contamination bygas-producing microorganisms, including, for example, E. coli, coliformbacteria, enterobacteria, Salmonellae, or the like. Culture devicesgenerally comprise a type of growth medium commonly used for biologicalgrowth and bacterial detection and enumeration. The invention, however,may also be applied with other types of growth media as discussedherein.

In any embodiment, the thin film culture device can have a front sidethat comprises a transparent film cover sheet and a back side comprisesa translucent substrate, such as a PETRIFILM E. coli/Coliform CountPlate, a PETRIFILM Coliform Count Plate, and a PETRIFILMEnterobacteriaceae Count Plate, for example. Without being bound bytheory, it is believed the combination of a relatively thin (e.g.,approximately 1-2 mm thick) culture medium disposed between atranslucent film and a transparent film provides optical conditions thatare beneficial for distinguishing gas-producing colonies according tothe present disclosure.

In order to improve the accuracy of automated counts of microbialcolonies on a culture device, various aspects of the methods of thepresent disclosure establish rules that can be applied during imageprocessing. In other words, the rules described in greater detail belowcan form part of a colony counting algorithm executed in system 20 or inan image-analysis system (not shown) that does not receive the imagesdirectly from an imaging device. The rules may be used individually orin any combination with other image analysis rules (the counting rulesdescribed in International Patent Publication No. WO 2005/062744),depending on the type of growth medium being scanned and the problemsthat may be encountered. Application of one or more of the countingrules can improve a biological scanning system such as system 20, forexample, by improving the accuracy of automated counts of microbialcolonies on a growth medium such as a thin film culture device or thelike.

FIG. 2 is a block diagram of a biological scanning system 30, which maycorrespond to system 20 (FIG. 1). System 30 includes an imaging device32 that generates one or more images of a growth medium and provides theimages to processor 34. Processor 34 is coupled to memory 36. Memory 36stores various processor-executable software instructions thatfacilitate image analysis of the images generated by imaging device 32.In particular, memory 36 stores one or more counting rules 37 which areapplied during image analysis to improve the accuracy of automatedcounts of microbial colonies on a culture device. Output device 38receives the results determined by processor 34 and provides the resultsto a user.

By way of example, imaging device 32 may comprise a 2-dimensionalmonochromatic camera for generating one or more images of a culturedevice. Various illuminators (not shown) may be used to illuminate thefront and back of culture device. For example, the illuminators canilluminate the culture device with one or more colors, and one or moreimages of the culture device can be generated by imaging device 32. Inaddition, a controller (not shown) can control a ratio of front-sideillumination to back side illumination for each image of the culturedevice. A non-limiting example of an imaging device that providesfront-side and back-side illumination, optionally with a plurality ofillumination colors, that can be used to image a thin film culturedevice is described in U.S. Pat. No. 8,094,916, which is incorporatedherein by reference in its entirety.

Methods of analyzing an image according to the present disclosureinvolve using at least one image of a culture device. Optionally, themethod may use two images; each image obtained using differentconditions to illuminate the culture device, to more-accuratelydistinguish gas-producing microbial colonies. A first image can beobtained while illuminating the “front side” of a culture device (i.e.,the side of the culture device facing the imaging device). The frontside of a thin film culture device is the side with the transparentcover sheet. A second image can be obtained while illuminating the “backside” of the culture device. The back side of a thin film culture deviceis the side opposite the cover sheet. In many PETRIFILM configured todetect gas-producing microbial colonies plates (e.g., PETRIFILM E.coli/Coliform Count Plates, PETRIFILM Coliform Count Plates, PETRIFILMRapid Coliform Count Plates, and PETRIFILM Enterobacteriaceae CountPlates), the back side of the thin film culture device comprises atranslucent polymeric film.

Illuminating the “front side” of a culture device can comprise exposingthe culture device to illumination coming from illuminators illuminatingthe front side of the device. In an embodiment, the first image can beobtained using 100% of the illumination coming from illuminatorsilluminating the front side of the culture device and 0% of theillumination coming from illuminators illuminating the back side of theculture device. In another embodiment, for example, a first image can beobtained using 80% of the illumination coming from illuminatorsilluminating the front side of the culture device and 20% of theillumination coming from illuminators illuminating the back side of theculture device. The ratio of front-side illumination to back-sideillumination can be selected to provide optimum contrast for aparticular type of nutrient medium in the culture device.

In any embodiment of the method, the first image is produced whileilluminating the device with a first ratio (e.g., 100%:0%) of front-sideillumination to back-side illumination and the second image is producedwhile illuminating the device with a second ratio (e.g., 0%:100%) offront-side illumination to back-side illumination that is lower than thefirst ratio. In any embodiment, the first ratio can be greater than 1:1.In any embodiment, the second ratio can be less than 1:1.

It should be noted that “first image”, as used herein, refers to animage that is obtained while the culture device receives illuminationprimarily from the front side of the plate and “second image”, as usedherein refers to an image that is obtained while the culture devicereceives illumination primarily from the back side of the plate. Animplied temporal order of obtaining the images is not intended by theuse of the terms “first image” and “second image”. Accordingly, a firstimage of a culture device can be obtained before or after a second imageof the culture device. In addition, one of the images (e.g., the firstimage or second image, respectively) does not need to be obtained by theimaging culture device immediately after obtaining the other image(e.g., the second image or first image, respectively). It is recommendedthe first and second images are obtained closely enough in time toobviate the possibility of significant biological changes (e.g., growthor enzyme activity) or physical changes (e.g., dehydration) occurringduring the intervening time between image acquisitions. Thus, in apreferred embodiment, the first image is obtained within about 30seconds of the time at which the second image is obtained.

A person having ordinary skill in the art will recognize that, in asystem wherein the imaging device is positioned facing the front side ofthe culture device and the illuminators are also positioned such thatthe illumination is directed at the front side of the culture device,the image produced by the imaging device substantially comprises lightthat is reflected from the culture device and the contents thereof. Inaddition, the person having ordinary skill in the art will alsorecognize that, in a system wherein the imaging device is positionedfacing the front side of the culture device and the illuminators arealso positioned such that the illumination is directed at the back sideof the culture device, the image produced by the imaging devicesubstantially comprises light that is transmitted by and/or refracted bythe culture device and the contents thereof.

Illuminating the “back side” of a culture device can comprise exposingthe culture device to illumination coming from illuminators illuminatingthe back side of the device. In an embodiment, the second image can beobtained using 100% of the illumination coming from illuminatorsilluminating the back side of the culture device and 0% of theillumination coming from illuminators illuminating the front side of theculture device. In another embodiment, for example, a second image canbe obtained using 80% of the illumination coming from illuminatorsilluminating the back side of the culture device and 20% of theillumination coming from illuminators illuminating the front side of theculture device. The ratio of front-side illumination to back-sideillumination can be selected to provide optimum contrast for aparticular type of nutrient medium in the culture device.

In any embodiment of the methods of the present disclosure, it may bedesirable to use only images obtained with back-side illumination orimages used with front-side illumination. As illustrated in FIGS. 6 and7 and discussed below, back-side illumination can provide particularlyenhanced contrast between the culture medium and a gas bubble (e.g., theperimeter of a gas bubble) in a thin film culture device.

The images are provided to processor 34 and may also be stored in memory36. In any case, the images are analyzed by applying counting rules 37in order to determine bacteria counts on the culture device. Theresolution of imaging device 32 may be approximately 155 pixels percentimeter. In that case, a one centimeter line in the image is 155pixels long.

Processor 34 may comprise a general-purpose microprocessor that executessoftware stored in memory 36. Alternatively, processor 34 may comprisean application specific integrated circuit (ASIC) or other specificallydesigned processor. In any case, processor 34 executes various countingrules 37 to improve the accuracy of automated counts of microbialcolonies on a culture device.

Memory 36 is one example, of a computer readable medium that storesprocessor executable software instructions applied by processor 34. Byway of example, memory 36 may comprise random access memory (RAM),read-only memory (ROM), non-volatile random access memory (NVRAM),electrically erasable programmable read-only memory (EEPROM), flashmemory, or the like. Counting rules 37 such as those described below,are stored in memory 36 and may form part of a larger software programused for image analysis.

Output device 38 typically comprises a display screen used tocommunicate results to a user. However, output device 38 could alsocomprise other types of devices such as a printer or the like. Outputdevice 38 may form part of a scanning unit, such as display (not shown),or may be external to the scanning unit, such as the display screen ofexternal computer 22 (FIG. 1).

FIG. 3 is a flow diagram illustrating a process of automated culturedevice analysis. As shown in FIG. 3, processor 34 receives one or moreimages of a culture device (step 41). Processor 34 invokes varioussoftware routines from memory 36 to identify the microbial colonies onthe culture device (step 42). For example, bacterial colonies may beidentified according to a characteristic color they produce afterreacting (i.e., directly or indirectly) with one or more indicatorcompounds in the nutrient medium. Other aspects of colony recognitionare discussed herein. The software executed by processor 34 can allowfor identification of the growth area on the culture device andautomated counting of bacterial colonies based on color changes in thegrowth area where the colonies have grown during incubation. Optionally,the processor can invoke a routine to count the identified microbialcolonies. Gas-producing colonies can be segregated into a count that isseparate from a count of colonies that do not produce gas in the culturedevice.

In accordance with a method of present disclosure, processor 34 appliesone or more rules to improve the accuracy of the count of microbialcolonies on the growth medium (step 43). The rules may be appliedindividually or various combinations of rules may be used, depending onthe type of culture device being analyzed. The rules may be individuallyinvoked from memory 36 or may form sub-routines of a larger imageanalysis software program. The rules may be applied individually orvarious sets of the rules may be applied. If a set of rules are used,then the order in which the rules are applied may be selected based onthe type of plate being scanned to obtain one or more image. Theselected order for application of the rules may affect the end result.Various subsets of the rules may also be applied in any order, and theselected order for a subset of rules may also affect the end result.

FIG. 4 shows one embodiment of a method 100 according to the presentdisclosure. The method comprises the step 51 of obtaining a first imagewhile illuminating the front side of a culture device and the step 52 ofobtaining a second image while illuminating the back side of the culturedevice. The front side and back side of the culture device can beilluminated with an imaging system as disclosed herein. The method 100further comprises the step 53 of analyzing the first image to identifythe location of microorganism colonies in the culture device. The method100 further comprises the step 54 of analyzing the second image toidentify the location of gas bubbles in the culture device.

Analyzing the second image to identify the location of a gas bubble cancomprise identifying a gas bubble as an object (in the image) that isdistinguishable from the culture medium in the culture device.Identifying the gas bubble as an object distinguishable from the culturemedium can be done by applying image analysis techniques disclosed byWeiss (U.S. Pat. No. 6,381,353). Analyzing the images can include RGB(red/green/blue) image processing algorithms. Alternatively, oradditionally, analyzing the images can include HSI (hue, saturation, andintensity), HSL (hue, saturation, and lightness), HSV (hue, saturation,and value) algorithms, or combinations thereof. Because the gas bubblesproduced by gas-producing colonies often displace some or all of theculture medium disposed between the cover sheet and the substrate of athin film culture device, a gas bubble can be detected as a region inthe image with substantially less color than the surrounding culturemedium and/or microbial colony. However, the unique configuration of thethin film culture devices permits an alternative approach to detect thepresence of a gas bubble in the image—detecting a relatively darkannulus surrounding the gas bubble.

Without being bound by theory, the edge of the gas bubble contacting theculture medium can serve as a lens to permit the transmission of lightthroughout the gas bubble. In addition, the edge can serve as a mirrorto reflect the image of the transmitted light. Thus, because the culturemedium and colonies are typically colored and the color is typicallydarker than the substrate forming the back of the thin film culturedevice, the transmitted (and reflected) image of the culture mediumand/or colony forms a relatively dark-colored annulus surrounding therelatively light-colored central portion of the gas bubbles. This darkannulus can be detected as a sharp color and/or brightness change thatis easily distinguishable from the culture medium and from microbialcolonies, which are typically darker than the culture medium, as shownin FIG. 6 and described in Example 1.

The first and second images are obtained so as to define objects in theimage in shades of at least one color. Thus, analyzing the first and/orsecond images to identify microorganism colonies in each image cancomprise identifying objects in the image as colonies according to imageanalysis methods that are well known in the art. For example, Weissdescribes techniques to identify objects (e.g., microbial colonies) inan image based upon one or more criteria including object size,visibility, color, surface quality, and shape (U.S. Pat. No. 6,243,486,which is incorporated herein by reference in its entirety). As discussedabove, a method to detect a microbial colony that reacts with anindicator compound comprising TTC can be configured to detect a shade ofthe color red and a method to detect a microbial colony that reacts withan indicator compound comprising5-bromo-4-chloro-3-indolyl-β-D-glucuronide can be configured to detect ashade of the color blue.

Analyzing the first and/or second images to identify microorganismcolonies in the image comprises identifying the location any coloniesdetected in the images. The location of a colony in the culture deviceis used to compare the location of the colony to a location of a gasbubble in the culture device. The location of a colony or a gas bubblecan be identified by X-Y coordinates in the image. Thus, in a preferredembodiment, both the first and second images are obtained without movingor otherwise handling the culture device after the first image isobtained but before the second image is obtained. Alternatively,registration landmarks (e.g., two or more corners of a PETRIFILM plate,or registration marks made on any culture device) can be used to orientthe images properly in order to determine and compare the location ofobjects (e.g., colonies, gas bubbles) in the first and second images.

After analyzing the first and second images to identify the location ofthe microorganism colonies and gas bubbles in the culture device, themethod 100 further comprises the step 55 of comparing the locations ofthe microorganism colonies and the gas bubbles. Comparing the locationscan comprise calculating the distance (e.g., in pixels) between aparticular microbial colony and one or more gas bubbles proximate thecolony. This distance can be used to determine whether the gas bubble isassociated with a particular colony and, thereby, conclude theparticular colony is a gas-producing microbial colony.

The Interpretation Guide provided by the manufacturer for use withPETRIFILM E. coli/Coliform Count Plates provides guidance regarding thedetermination whether a gas bubble is associated with a particularcolony. In part, the guidance relates to the proximity of the gas bubbleto a microbial colony. In general, a gas bubble contacting a particularmicrobial colony is regarded as being associated with (i.e., producedby) the colony. In addition, a gas bubble that is located within adistance equal to about three colony diameters is regarded as beingassociated with (i.e., produced by) the colony. Thus, analyzing an image(e.g., the first image or second image disclosed herein) to identify apresence or a location of a gas-producing microorganism according to thepresent disclosure comprises analyzing the image to identify thepresence or location of one or more gas bubbles. The location of a gasbubble relative to any proximate microbial colonies is used to determinewhether the gas bubble produced by the microorganisms that form thecolony.

Analyzing an image (e.g., the first image or second image disclosedherein) to identify a presence or a location of one or more gas bubblefurther can comprise analyzing the image to determine a size parameter(e.g., the radius, the diameter, and/or the area) of the one or more gasbubble in the image. The size parameter can be determined by calculatinga number of pixels in the radius, diameter, or area of the gas bubble,for example. When a plurality of gas bubbles are observed in an image,the size parameters for each of the plurality of gas bubbles can becompared, for example, in a histogram. The histogram can show thedistribution of sizes (e.g., radii, diameters, or areas) of gas bubblesfound in the image. In addition, the histogram may show gaps in the sizedistribution of the gas bubbles. These gaps can be used to differentiateabiogenic gas bubbles from biogenic gas bubbles.

International Patent Publication No. WO2012/012104, which isincorporated herein by reference in its entirety, describes the presenceof small, abiogenic gas bubbles that may be observed in a thin filmculture device. The application further discloses the diminution ordisappearance of the abiogenic gas bubbles within a region surrounding amicrobial colony as the microbial colony develops. The abiogenic bubblesand the region (i.e., the region devoid of abiogenic gas bubbles)surrounding a microbial colony can be clearly seen in FIG. 6, describedbelow. Thus, any embodiment of a method of the present disclosure mayfurther comprise calculating a number of gas bubbles in a regionsurrounding a colony. The area of the region surrounding the colony canbe less than or equal to about five times the area of the colony, forexample. The method optionally may comprise comparing a number ofobservable gas bubbles within the region proximate a microbial colony toa number of observable gas bubbles in another region (e.g., a “control”region) of the nutrient medium in the culture device that does notcomprise a colony. When this comparison is made, if the number ofobservable gas bubbles in the region proximate the microbial colony issubstantially lower than the number of observable gas bubbles in thecontrol region, the presence of a first gas bubble proximate themicrobial colony, regardless of the size of the first gas bubble, mayindicate the microbial colony is a gas-producing microbial colony.

In any embodiment, the method of the present disclosure further cancomprise comparing the size parameter of a first gas bubble to the sizeparameter of a second gas bubble. When a plurality of gas bubbles ispresent in the image, comparing the size parameter of the first gasbubble to the size parameter of a second gas bubble can comprisecreating a histogram of the size parameter values for each of theplurality of gas bubbles. The distribution of size parameter values(e.g., radii, diameters, or areas) may reveal clusters ofsimilarly-sized gas bubbles. The clusters can be divided into one ormore groups.

The histogram can be used to determine a threshold size parameter value(e.g., for the bubble radius, diameter, or area) or a size parameterrange that is used to distinguish a first gas bubble that is produced bya gas-producing microorganism from a second gas bubble that is notproduced by a gas-producing microorganism. Advantageously, comparing thesize of a plurality of gas bubbles observed in the culture device can beused to distinguish abiogenic gas bubbles, which tend to have arelatively uniform size, from gas bubbles produced by a microbialcolony, which are frequently larger than the abiogenic gas bubbles.

The Interpretation Guide provided by the manufacturer for use withPETRIFILM E. coli/Coliform Count Plates provides additional guidanceregarding the determination whether a gas bubble is associated with aparticular colony. In general, when a gas bubble is proximate aparticular microbial colony and the gas bubble has a diameter that isabout equal to or greater than the diameter of the proximate colony, thegas bubble is regarded as being associated with (i.e., produced by) thecolony. However, a person having ordinary skill in the art willrecognize there may be instances (e.g., when the sample and/or culturedevice comprises a nutrient that inhibits microbial gas production)wherein a gas bubble associated with a gas-producing colony may have asize parameter value (e.g., diameter) that is smaller than gas-producingmicrobial colony with which it is associated.

FIG. 5 is a flow diagram illustrating the rule for differentiatingcolonies into a plurality of colony types according to the presentdisclosure. As illustrated in FIG. 2, processor 34 invokes softwarestored in memory 36 to identify and map the location of colonies and gasbubbles in the first and second images (step 61). In particular,processor 34 determines whether a colony that is identified and locatedusing the first image maps to a location proximate a gas bubble that isidentified and located using the second image (step 62). If coloniesthat are proximate gas bubbles are found when analyzing the first andsecond images, the processor 34 applies a proximity factor to decidewhether the gas bubble is associated with (i.e., produced by) theproximate colony. If the gas bubble is within a predetermined distanceof the colony (e.g., within the equivalent of about three colonydiameters), as discussed above, the colony in the culture device iscounted as a first type (e.g., a “Gas-Producing”; see step 63, FIG. 5).If the gas is not within a predetermined distance of the colony (e.g.,further away than the equivalent of about three colony diameters), asdiscussed above, the colony in the culture device is counted as a secondtype (e.g., a “Non-Gas-Producing”; see step 64, FIG. 5).

Optionally, additional steps (not shown) can be added to the countingrule illustrated in FIG. 5. The additional step can be inserted into thesequence of steps at any point after step 61 and before step 63 or step64. A first additional step relates the map location of each gas bubblewith the size (e.g., diameter or area) of the gas bubble in the image. Asecond additional step compares the size of the gas bubble to apredetermined size value (or a range of predetermined size values) todetermine whether the size of a particular gas bubble is less than,greater than or equal to the predetermined size value or to determinewhether the size of the particular gas bubble is within thepredetermined range. Thus, a counting rule comprising the additionalsteps utilize two parameters (i.e., size of a gas bubble and theproximity of the gas bubble to a colony) associated with each gasbubble) to make a determination whether the gas bubble is associatedwith microbial activity (i.e. determine whether a microbial colonyshould be counted as a gas-producing microbial colony).

In some embodiments of the method, identifying a microbial colony as agas-producing microbial colony can permit the operator to identify aparticular classification or group to which the microorganism in thecolony belongs. For example, if the culture medium comprises nutrientsand selective agents that facilitate the growth of Gram-negative entericbacteria and the fermentable nutrient in the culture medium is lactose,the microorganisms can be presumptively identified as belonging to agroup of bacteria known as coliform bacteria. In another example, if theculture medium comprises nutrients and selective agents that facilitatethe growth of Gram-negative enteric bacteria and the fermentablenutrient in the culture medium is glucose, the microorganisms can bepresumptively identified as belonging to a family of bacteria known asEnterobacteriaceae.

FIG. 6 shows a black-and-white image of a portion of the growth area ofa thin film culture device. The image was obtained while illuminatingonly the back side of the culture device, as described in Example 1.Within the growth area are a plurality of microbial colonies (71-73).Also shown in FIG. 6 is a plurality of gas bubbles (81-84). It can beobserved that gas bubbles 81-83 have an area that is larger than atleast one microbial colony (e.g., colony 71) that is proximate (i.e.,within a distance that is less than or equal to about three colonydiameters) the gas bubbles. Thus, according to the method of the presentdisclosure, all three of the gas bubbles (bubbles 81-83) would beconsidered “biogenic” gas bubbles (i.e., they were produced bymicroorganisms in the sample). In contrast, there are numerous, verysmall gas bubbles 84 distributed throughout the culture medium in thegrowth area of the culture device. Because these bubbles (i.e., gasbubbles 84) are evident in the culture device shortly after it isinoculated (i.e., hours before microbial colonies appear in the culturedevice) and because the area (volume) of the bubbles does not increase(e.g., generally, they remain substantially smaller than a typicalmicrobial colony diameter) after prolonged incubation, they areconsidered “abiogenic” gas bubbles (i.e., they are not produced bymicroorganisms in the sample), as discussed above. Also shown in FIG. 6is a path 90 of a line scan of a portion of the growth area of theculture device. Path 90 traverses a portion of the image that includesgas bubble 82.

When a larger portion of the image shown in FIG. 6 was analyzed todetermine the area of the gas bubbles, it was observed that, for thisimage, the average area of approximately ninety of the abiogenic gasbubbles (i.e., gas bubbles 84) was less than or equal to 50 pixels(i.e., an area of about 50 pixels represented the upper size limit ofthe relatively small, abiogenic gas bubbles. In contrast, the individualarea of each of the biogenic (i.e., associated with microbial activity)gas bubbles 81-83 in the two-dimensional image of FIG. 6 wasapproximately 625 pixels, 1125 pixels, and 875 pixels, respectively.Thus, the gap in size between the relatively tight cluster of abiogenicgas bubbles (i.e., those gas bubbles having an area less than or equalto about 50 pixels) and the relatively loose cluster of biogenic gasbubbles (i.e., those gas bubbles having an area greater than or equal toabout 500 pixels) can be used to set a lower threshold value (e.g.,about 75 pixels, about 100 pixels, about 200 pixels, or about 250pixels) that can be used to designate biogenic gas bubbles in aparticular image. In any embodiment, this threshold can be set using adynamic calculation (i.e., calculated based upon the bubble populationof a particular image of a particular culture device), which bases thethreshold on a set percentage (e.g., about 125%, about 200%, about 300%,about 400%) of the maximum size parameter value of the abiogenic gasbubble group.

Thus, according to a method of the present disclosure, the gas bubblescan be classified into at least two group; a first group having atwo-dimensional area of about 50 pixels or less and a second grouphaving a two-dimensional area greater than about 50 pixels. Accordingly,the gas bubbles classified (e.g., according to size) into the firstgroup can be identified as suspect abiogenic gas bubbles (i.e., probablynot microorganism-associated). Conversely, gas bubbles classified (e.g.,according to size) into the second group can be identified as suspectbiogenic gas bubbles (i.e., probably microorganism-associated). In anyembodiment of the method, a lower size limit can be assigned for thesecond group. For example, the lower size limit for the second group maybe a size parameter value that is at least 50 percent larger, at least100% larger, or at least 150% larger than the estimated upper size limitof the abiogenic gas bubbles. According to the method, any culturedevice having a gas bubble that has a size parameter value fallingbetween the upper size limit of the first group and the lower size limitof the second group could optionally be flagged for review by atechnician.

In some embodiments of the method, an upper size limit for the suspectbiogenic gas bubbles can be assigned (e.g., by running control culturedevices to establish the maximum size of biogenic gas bubbles for givenmicroorganisms). In these embodiments, a gas bubble exceeding the uppersize limit of the second group would be classified into a third group.The third group includes relatively large abiogenic gas bubbles whichmay have been introduced into the culture device during inoculation, forexample, which may indicate operator usage problems. When a bubbleexceeding the upper size limit is observed in an image, an advisorymessage may be posted to the operator.

FIG. 7 shows a black and white image of the same portion of the thinfilm culture device shown in FIG. 6. The image was obtained whileilluminating only the front side of the culture device, as described inExample 1. Although the biogenic gas bubbles (i.e., gas bubbles 81-83 ofFIG. 6) are visible, the contrast between the culture medium and thebiogenic gas bubbles is significantly lower. In addition, the outer edgeof the gas bubbles is not as clearly delineated in the image of FIG. 7as it is in the back-lit image of FIG. 6. Also shown in FIG. 7 is a path91 of a line scan of the portion of the growth area shown in FIG. 6.Path 91 corresponds to the same pixels as those in path 90 of FIG. 6.

In order to identify the presence and location of colonies in a back-litimage and to identify the presence and location of microbial colonies ina front-lit image, image-analysis algorithms often analyze the pixels inthe digital image line-by-line, comparing the color hue and/or colorintensity of a first pixel or first group of pixels to the color hueand/or color intensity of a second pixel proximate the first pixel orsecond group of pixels proximate the first group of pixels. This type ofcomparison permits the algorithm to recognize color and/or intensityshifts that may indicate the edge of a microbial colony, gas bubble, orother object in the image. FIG. 8 shows a graph of the transmitted colorintensities for red, green, and blue from pixels along the line 90 inthe back-lit image of FIG. 6. The sharp demarcation of the gas bubbles,as evidenced by dark annulus associated with the opposite edges of theperimeter of the gas bubble can be seen in the graph as sharp negativepeaks (A and B, respectively). FIG. 9 shows a graph of the reflectedpixel intensities for red, green, and blue obtained from pixels alongthe line 91 in the front-lit image of FIG. 7.

In addition to providing a method of analyzing an image to identify thepresence and individual size of gas bubbles in a thin film culturedevice, the present disclosure also provides a method of analyzing animage to identify the two dimensional context of a particular gas bubble(e.g., a “first” gas bubble) in a thin film culture device.“Two-dimensional context” of a first gas bubble refers to the presenceor absence of other gas bubbles within a predefined area proximate thefirst gas bubble. The presence and number of proximate gas bubbles canbe used to determine whether the first gas bubble is a suspect biogenicgas bubble (i.e., probably associated with microbial activity) or is asuspect abiogenic gas bubble (i.e. probably not associate with microbialactivity).

By way of example, FIG. 10A shows an image of a larger portion of thethin film culture device shown in the image of FIG. 6. In FIG. 10A,microbial colonies 71, 72, 73, 74, and 75 are evident by theirinteraction with the chromogenic indicators present in the culturemedium. Also evident in FIG. 10A are abiogenic gas bubbles 84 andbiogenic gas bubbles 81, 82, 83, 85, 86, and 87. In one aspect, thebiogenic gas bubbles (81-83 and 85-87) can be identified as biogenic onthe basis of their respective size and/or proximity to a microbialcolony, as described herein. In another aspect, the biogenic gas bubblescan be identified on the basis of their two-dimensional context. FIG.10A shows that, although abiogenic gas bubbles are substantiallyuniformly dispersed in a region of the culture medium that does notinclude a microbial colony (see FIG. 10B), the size of the abiogenic gasbubbles is substantially smaller or the abiogenic gas bubbles aresubstantially absent in regions (e.g., regions 101, 102, and 103 of FIG.10A) of the culture medium that include one or more microbial colony.This observed phenomenon can be used to identify a suspect biogenic gasbubble without the need to observe a microbial colony proximate theparticular suspect gas bubble.

One technique for analyzing the two-dimensional context of a particulargas bubble in an image of a thin film culture device is to divide theimage of the growth are of the culture device into a plurality ofsubdivisions, each subdivision having a uniform, predetermined size andshape and to count the number of gas bubbles in each subdivision. Thiscan be done, for example, by masking techniques that are known in theart of image analysis. FIGS. 10B-10D show the image of FIG. 10A withdenoted areas (areas A1, A2, and A3, respectively) enclosed by a mask orframe of a uniform size and shape. It can be observed that area A1,which does not encompass any microbial colonies, comprises approximatelythirty-four relatively small, substantially uniformly-distributedabiogenic gas bubbles. In contrast, area A2 (FIG. 10C), whichencompasses at least two microbial colonies, comprises a total ofapproximately seven gas bubbles that are not substantiallyuniformly-distributed in area A2. In addition, area A3 (FIG. 10D), whichencompasses one microbial colony, comprises a total of about twenty-fiverelatively small, substantially uniformly-distributed abiogenic gasbubbles.

According to a method of the present disclosure, analyzing an image of athin film culture device can comprise analyzing a plurality of areas todetect the number of gas bubbles present in the area. In any embodiment,two or more of the areas can overlap. In an embodiment, the mask used toanalyze each area can be rastered across the image line by line toobserve differences between neighboring areas and, thereby identifyparticular areas of interest (within the image) that might include amicrobial colony. The areas of interest (e.g., regions 101, 102, and 103of FIG. 10A) can be identified by the lower number of gas bubbles in thearea of interest relative to other areas (e.g., similar-sized areas)within the image.

According to a method of the present disclosure, analyzing the imagefurther can comprise identifying a suspect region (e.g., one or more ofthe aforementioned areas of interest) of the image, wherein the suspectregion has a substantially reduced number of gas bubbles relative toanother portion of the image. In these instances, a presence of a gasbubble in a portion of the suspect region (e.g., a central portion ofthe suspect region) may indicate the presence of a biogenic gas bubbleassociated with microbial activity. When this circumstance is detected,the size of the gas bubble detected in the suspect region may confirmthat the gas bubble in the suspect region is a biogenic gas bubble. Ifthe size of the gas bubble in the suspect region is larger thanabiogenic gas bubbles detected in other regions of the image, this isstrongly indicative the gas bubble is biogenic. Regardless of its size,a gas bubble proximate a microbial colony that is located in a suspectregion (i.e., a region of the image with significantly fewer gas bubblesthat other regions of the culture medium in the image) may be a biogenicgas bubble and the culture device may be reported as positive forgas-producing microorganisms. Alternatively or additionally, the imagemay be flagged for review by an operator.

The use of a scanning system and/or image-analyzing system with acounting rule for differentiating gas bubbles and/or microbial coloniesin an image of a thin film culture device has been described. Thecounting rule can be used in a scanning system to improve the accuracyof automated counts of microbial colonies on a culture device.

The aforementioned techniques for analyzing an image of a thin filmculture device can be used in methods for detecting a presence or anabsence of a microorganism in a sample inoculated into the thin filmculture device. In one aspect, the method comprises analyzing an imageof the growth area of a thin film culture device to detect gas bubblesand classifying a plurality of the gas bubbles, wherein classifying theplurality of gas bubbles comprises assigning each gas bubble to a firstgroup or a second group according to a size parameter associated witheach of the plurality of gas bubbles. The image of the growth area canbe analyzed to detect the gas bubbles as described herein. The bubblescan be classified according to a size parameter and an upper size limitand/or lower size limit for each group can be assigned as disclosedherein. In any embodiment, classifying the gas bubbles into a firstgroup and a second group can comprise classifying a first subset of gasbubbles into a first suspect abiogenic bubble group. In theseembodiments, the method further comprises assigning a size parametervalue upper limit for the first suspect abiogenic bubble group, asdescribed herein. In any embodiment, classifying the gas bubbles into afirst group and a second group can comprise classifying a second subsetof gas bubbles into a suspect biogenic bubble group. In theseembodiments, the method further comprises assigning a size parametervalue lower limit for the suspect biogenic bubble group, as describedherein.

In another aspect, the method comprises analyzing a first area of animage of a growth area of a thin film culture device to detect a firstnumber of gas bubbles in the first area, analyzing a second area of theimage to detect a second number of gas bubbles in the second area, andcomparing the first number of gas bubbles to the second number of gasbubbles. Analyzing the first and second areas can comprise using animage mask to count the number of gas bubbles within the first andsecond areas, wherein the image mask defines a specified magnitude andshape of the area, as described herein. In any embodiment, the methodfurther can comprise analyzing a third area of the image to detect athird number of gas bubbles in the third area, and comparing the firstnumber of gas bubbles or second number of gas bubbles to the thirdnumber of gas bubbles. The first area, second area, and/or third areacan be spaced apart from each other or at least two of the areas maypartially overlap.

In yet another aspect two or more of the methods described herein may becombined into a single method to detect a presence or an absence of amicroorganism in a thin film culture device. For example, the method cancomprise analyzing a first area of an image of a growth area of a thinfilm culture device to detect a first number of gas bubbles in the firstarea, analyzing a second area of the image to detect a second number ofgas bubbles in the second area, comparing the first number of gasbubbles to the second number of gas bubbles, analyzing the image todetect gas bubbles in the growth area of the culture device, andclassifying a plurality of the gas bubbles, wherein classifying theplurality of gas bubbles comprises assigning each gas bubble to a firstgroup or a second group according to a size parameter associated witheach of the plurality of gas bubbles. In some embodiments, the methodfurther can comprise determining whether a gas bubble in any of thefirst, second, or third areas is assigned to the first group or thesecond group. Advantageously, in this embodiment, the size of aparticular gas bubble is analyzed in a context that accounts for thenumber and size of gas bubbles in its proximity, thereby using twoseparate criteria to confirm whether the particular gas bubble isbiogenic or abiogenic.

The techniques have been described as being software-implemented. Inthat case, a computer readable medium stores processor executableinstructions that embody one or more of the rules described above. Forexample, the computer readable medium may comprise non-transitorycomputer readable media such as random access memory (RAM), read-onlymemory (ROM), non-volatile random access memory (NVRAM), electricallyerasable programmable read-only memory (EEPROM), flash memory, or thelike. The computer readable medium may also comprise a non-volatilememory such as a CD-ROM used to deliver the software to customers. Also,the computer readable medium may comprise an electromagnetic carrierwave, e.g., for delivering the software over a network such as theinternet.

The same techniques, however, may also be implemented in hardware.Example hardware implementations include implementations within anapplication specific integrated circuit (ASIC), a field programmablegate array (FPGA), specifically designed hardware components, or anycombination thereof. In addition, one or more of the techniquesdescribed herein may be partially executed in hardware, software orfirmware.

Thus, the present disclosure provides a computer readable mediumcontaining computer readable instructions for detecting a presence or anabsence of a microorganism in a thin film culture device. In one aspect,the computer readable instructions, when executed by a processor causean image-analyzing system comprising the processor to analyze a firstimage of a thin film culture device, the culture device having a frontside and a back side opposite the front side; wherein the first image isproduced while providing illumination to the front side of the device asdescribed herein. Analyzing the first image comprises identifying amicroorganism colony at a first location in the culture device. Thecomputer readable instructions, when executed by the processor, furthercause the processor to analyze a second image of the thin film culturedevice; wherein the second image is produced while providingillumination to the back side of the device. Analyzing the second imagecomprises identifying a gas bubble at a second location in the culturedevice, as described herein. The computer readable instructions, whenexecuted by the processor, further cause the processor to determinewhether the first location is within a predetermined distance from thesecond location.

In another aspect, the computer readable instructions, when executed bya processor cause an image-analyzing system comprising the processor toanalyze an image of the growth area of a thin film culture device todetect gas bubbles and to classify a plurality of the gas bubbles.Classifying the plurality of gas bubbles comprises assigning each gasbubble to a first group or a second group according to a size parameterassociated with each of the plurality of gas bubbles, as describedherein. In any embodiment, classifying the gas bubbles into a firstgroup and a second group can comprise classifying a first subset of gasbubbles into a first suspect abiogenic bubble group, wherein thecomputer readable instructions further cause the processor to assign asize parameter value upper limit for the first suspect abiogenic bubblegroup. In any embodiment, classifying the gas bubbles into a first groupand a second group can comprise classifying a second subset of gasbubbles into a suspect biogenic bubble group, wherein the computerreadable instructions further cause the processor to assign a sizeparameter value lower limit for the suspect biogenic bubble group.

In another aspect, computer readable instructions, when executed by aprocessor cause an image-analyzing system comprising the processor toanalyze a first area of an image of a growth area of a thin film culturedevice to detect a first number of gas bubbles in the first area, toanalyze a second area of the image to detect a second number of gasbubbles in the second area, and to compare the first number of gasbubbles to the second number of gas bubbles. In any embodiment, thecomputer readable instructions further cause the processor to analyze athird area of the image to detect a third number of gas bubbles in thethird area and to compare the first number of gas bubbles or secondnumber of gas bubbles to the third number of gas bubbles. In anyembodiment, the computer readable instructions further cause theprocessor to analyze the image to detect gas bubbles in the growth areaof the culture device and to classify a plurality of the gas bubbles,wherein classifying the plurality of gas bubbles comprises assigningeach gas bubble to a first group or a second group according to a sizeparameter associated with each of the plurality of gas bubbles, asdescribed herein. In some embodiments, the computer readableinstructions further cause the processor to determine whether a gasbubble in any of the first, second, or third areas is assigned to thefirst group or the second group.

In any embodiment, a computer readable medium comprising computerreadable instructions may comprise instructions that include two or moreof the methods described herein. The two or more methods can be usedindividually, or in combination, to improve the accuracy of thedetection of a gas-producing microbial colony.

Embodiments

Embodiment A is a method, comprising:

using an imaging device to produce a first image of a thin film culturedevice, the culture device having a front side having a transparent filmcover sheet and a back side having a translucent substrate;

-   -   wherein the first image is produced while providing illumination        to the front side of the device;    -   wherein the culture device comprises an indicator compound that        is converted by a microorganism to a first product that is        observable by a first color;    -   wherein the culture device comprises a nutrient that can be        converted to by a first type of microorganism to a gas;

using the imaging device to produce a second image of the thin filmculture device, wherein the second image is produced while providingillumination to the back side of the device;

analyzing the first image to identify a microorganism colony at a firstlocation in the culture device;

analyzing the second image to identify a first gas bubble at a secondlocation in the culture device; and

determining whether the first location is within a predetermineddistance from the second location.

Embodiment B is the method of Embodiment A, wherein the first image isproduced while illuminating the device with a first ratio of front-sideillumination to back-side illumination, wherein the second image isproduced while illuminating the device with a second ratio of front-sideillumination to back-side illumination that is lower than the firstratio.

Embodiment C is the method of Embodiment B, wherein the first ratio isabout 100%:0%.

Embodiment D is the method of Embodiment B or Embodiment C, wherein thesecond ratio is about 0%:100%.

Embodiment E is the method of any one of the preceding Embodiments,wherein the first gas bubble comprises a first perimeter, whereinanalyzing the second image to identify a first gas bubble comprisesidentifying a dark annulus associated with the first perimeter.

Embodiment F is the method of any one of the preceding Embodiments,wherein analyzing the second image to identify a first gas bubblecomprises calculating a size parameter of the first gas bubble.

Embodiment G is the method of Embodiment F, wherein the size parameteris a radius, a diameter, or an area.

Embodiment H is the method of any one of the preceding Embodiments,wherein analyzing the second image to identify a first gas bubblecomprises analyzing a first predetermined region surrounding the firstgas bubble to detect a second gas bubble.

Embodiment I is the method of Embodiment H, wherein the firstpredetermined region comprises a microbial colony, wherein the methodfurther comprises comparing a number of gas bubbles in the firstpredetermined region to a number of gas bubbles in a secondpredetermined region, wherein the second predetermined region does notcomprise a microbial colony.

Embodiment J is the method of any one of Embodiments F through I,further comprising comparing the size parameter of the first gas bubbleto the size parameter of a second gas bubble.

Embodiment K is the method of any one of the preceding Embodiments,further comprising using the first image to count a number ofmicroorganism colonies in the culture device.

Embodiment L is the method of Embodiment K, further comprising using thefirst and second images to count a number of microorganism colonies thatconvert the nutrient to a gas.

Embodiment M is the method of any one of the preceding Embodiments,further comprising using the first and second images to count a numberof microorganism colonies that don't convert the nutrient to a gas.

Embodiment N is the method of any one of the preceding Embodiments,wherein the indicator compound comprises a tetrazolium dye.

Embodiment O is the method of any one of the preceding Embodiments,wherein the nutrient comprises a carbohydrate.

Embodiment P is the method of Embodiment O, wherein the carbohydrate isselected from the group consisting of glucose, sucrose, lactose, or acombination of any two or more of the foregoing carbohydrates.

Embodiment Q is a computer readable medium comprising computer readableinstructions that, when executed by a processor, cause animage-analyzing system comprising the processor to:

analyze a first image of a thin film culture device, the culture devicehaving a front side and a back side opposite the front side;

-   -   wherein the first image is produced while providing illumination        to the front side of the device;    -   wherein analyzing the first image comprises identifying a        microorganism colony at a first location in the culture device;

analyze a second image of the thin film culture device;

-   -   wherein the second image is produced while providing        illumination to the back side of the device;    -   wherein analyzing the second image comprises identifying a gas        bubble at a second location in the culture device; and

determine whether the first location is within a predetermined distancefrom the second location.

Embodiment R is the computer readable medium of Embodiment Q, whereinanalyzing the second image to identify a second location of a gas bubblecomprises identifying a dark annulus surrounding the gas bubble.

Embodiment S is the computer readable medium of Embodiment Q orEmbodiment R, further comprising computer readable instructions that,when executed in the processor, cause the system to use the first imageto count a number of microorganism colonies in the culture device.

Embodiment T is the computer readable medium of Embodiment Q orEmbodiment R, further comprising computer readable instructions that,when executed in the processor, cause the system to use the first andsecond images to count a number of gas-producing microorganism coloniesin the culture device.

Embodiment U is a method, comprising:

analyzing an image of the growth area of a thin film culture device todetect gas bubbles; and

classifying a plurality of the gas bubbles, wherein classifying theplurality of gas bubbles comprises assigning each gas bubble to a firstgroup or a second group according to a size parameter associated witheach of the plurality of gas bubbles.

Embodiment V is the method of Embodiment T, wherein classifying the gasbubbles into a first group and a second group comprises classifying afirst subset of gas bubbles into a first suspect abiogenic bubble group,wherein the method further comprises assigning a size parameter valueupper limit for the first suspect abiogenic bubble group.

Embodiment W is the method of Embodiment U or Embodiment V, whereinclassifying the gas bubbles into a first group and a second groupcomprises classifying a second subset of gas bubbles into a suspectbiogenic bubble group, wherein the method further comprises assigning asize parameter value lower limit for the suspect biogenic bubble group.

Embodiment X is the method of Embodiment W, wherein the size parametervalue lower limit for the suspect biogenic bubble group is at leastabout two times larger than the size parameter value upper limit for thefirst suspect abiogenic bubble group.

Embodiment Y is a computer readable medium comprising computer readableinstructions that, when executed by a processor, cause animage-analyzing system comprising the processor to:

analyze an image of the growth area of a thin film culture device todetect gas bubbles; and

classify a plurality of the gas bubbles, wherein classifying theplurality of gas bubbles comprises assigning each gas bubble to a firstgroup or a second group according to a size parameter associated witheach of the plurality of gas bubbles.

Embodiment Z is the computer readable medium of Embodiment Y, whereinclassifying the gas bubbles into a first group and a second groupcomprises classifying a first subset of gas bubbles into a first suspectabiogenic bubble group, wherein the computer readable instructionsfurther cause the processor to assign a size parameter value upper limitfor the first suspect abiogenic bubble group.

Embodiment AA is the computer readable medium of Embodiment Y orEmbodiment Z, wherein classifying the gas bubbles into a first group anda second group comprises classifying a second subset of gas bubbles intoa suspect biogenic bubble group, wherein the computer readableinstructions further cause the processor to assign a size parametervalue lower limit for the suspect biogenic bubble group.

Embodiment BB is a method, comprising:

analyzing a first area of an image of a growth area of a thin filmculture device to detect a first number of gas bubbles in the firstarea;

analyzing a second area of the image to detect a second number of gasbubbles in the second area; and

comparing the first number of gas bubbles to the second number of gasbubbles.

Embodiment CC is the method of Embodiment BB, further comprising:

analyzing a third area of the image to detect a third number of gasbubbles in the third area; and comparing the first number of gas bubblesor second number of gas bubbles to the third number of gas bubbles.

Embodiment DD is the method of Embodiment BB or Embodiment CC, furthercomprising:

analyzing the image to detect gas bubbles in the growth area of theculture device; and

classifying a plurality of the gas bubbles, wherein classifying theplurality of gas bubbles comprises assigning each gas bubble to a firstgroup or a second group according to a size parameter associated witheach of the plurality of gas bubbles.

Embodiment EE is the method of Embodiment DD, further comprising:

determining whether a gas bubble in any of the first, second, or thirdareas is assigned to the first group or the second group.

Embodiment FF is a computer readable medium comprising computer readableinstructions that,

when executed by a processor, cause an image-analyzing system comprisingthe processor to:

analyze a first area of an image of a growth area of a thin film culturedevice to detect a first number of gas bubbles in the first area;

analyze a second area of the image to detect a second number of gasbubbles in the second area; and

compare the first number of gas bubbles to the second number of gasbubbles.

Embodiment GG is the computer readable medium of Embodiment FF, whereinthe computer readable instructions further cause the processor to:

analyze a third area of the image to detect a third number of gasbubbles in the third area; and

compare the first number of gas bubbles or second number of gas bubblesto the third number of gas bubbles.

Embodiment HH is the computer readable medium of Embodiment FF orEmbodiment GG,

wherein the computer readable instructions further cause the processorto:

analyze the image to detect gas bubbles in the growth area of theculture device; and

classify a plurality of the gas bubbles, wherein classifying theplurality of gas bubbles comprises assigning each gas bubble to a firstgroup or a second group according to a size parameter associated witheach of the plurality of gas bubbles.

Embodiment II is the computer readable medium of Embodiment HH, whereinthe computer readable instructions further cause the processor todetermine whether a gas bubble in any of the first, second, or thirdareas is assigned to the first group or the second group.

EXAMPLES Example 1 Method for Detecting Gas-Producing Colonies

Tryptic Soy Broth (TSB, Catalog #K89) was obtained from HardyDiagnostics (Santa Maria, Calif.). Microbial strains E. coli (ATCC25922), E. coli (3M-FR4), Salmonella enterica (ATCC 51812) andEnterobacter amnigenus (ATCC 51898) were obtained from MicrobiologicsInc (St Cloud, Minn.). An overnight TSB culture was prepared for eachmicrobial strain. Thin film culture devices (3M PETRIFILM E.Coli/Coliform Count (EC) Plates) and Butterfield's Phosphate Buffer wereboth obtained from the 3M Company (St. Paul, Minn.).

Dilutions from overnight cultures of each strain were prepared inButterfield's Phosphate Buffer to yield approximately 25 colony-formingunits (CFU) per mL. The 3M PETRIFILM plates were inoculated by liftingthe transparent film cover sheet, pipetting 1mL of the diluted sample inthe center of the coated bottom film, and replacing the cover sheet. Thesample was uniformly spread to the desired surface area (approximately20 cm²) using the spreading device provided by the manufacturer (3M).Inoculated plates were incubated at 35° C. for 24 hours.

The colonies on the PETRIFILM culture plate were imaged and identifiedusing a culture device imaging system. The imaging system contained acentrally positioned glass platen (White Flashed Opal Glass) that servedas a platform for placement of the culture plate. The culture plate wasilluminated using two separate sets of light emitting diodes (each setcontaining two red LEDs, two green LEDs, and two blue LEDs) One set waspositioned above to the left (relative to the longitudinal dimension) ofthe culture plate and the other set was positioned above and to theright (relative to the longitudinal dimension of the culture plate).Light from the LEDs positioned above the culture device was directedaway from the culture device and into a light-diffusing reflectivesurface, which directed a substantially uniform illumination pattern onthe front side of the culture plate. Similarly, the culture plate wasilluminated on the back side using two separate sets of light emittingdiodes (each set containing two red LEDs, two green LEDs, and two blueLEDs). One set was positioned below and to the left (relative to thelongitudinal dimension) of the culture plate and the other set waspositioned below and to the right (relative to the longitudinaldimension of the culture plate). Light from the LEDs positioned belowthe culture device was directed away from the culture device and into alight-diffusing reflective surface, which directed a substantiallyuniform illumination pattern onto the back side of the glass platen(described above) and created a uniform illumination pattern on the backside of the culture plate.

An Aptina Model MT9P031 CMOS imaging sensor (Aptina Imaging, San Jose,Calif.) was orthogonally-positioned above the platform and positioned totake images of the culture plate. The imaging sensor and platform wereadjusted so that the culture plate was positioned within the focal planeof the sensor. The culture plate was oriented on the platform so thatfront side (transparent film side) of the culture plate faced theimaging sensor. A black cover was used to isolate the imaging devicefrom room light. The image exposures were selected so that, in theacquired images, less than about 10% of the pixels in a histogram of allof the image pixels were saturated. A first image was taken using onlyillumination from the front side of the culture plate and a second imagewas taken using only illumination from the back side of the cultureplate. Both images were taken with the culture plate being maintained inexactly the same position on the platform (i.e., the plates were notmoved from the platform until both images were acquired). This allowedfor the identification of coincidental colonies in the two images bymatching the corresponding X-Y coordinate positions.

The two images were analyzed using ImagePro Plus software (MediaCybernetics, Rockville, Md.). The size of an individual colony wasdetermined from the first culture plate image (100% front-sideillumination). The imaging program analyzed for changes in red, green,and blue pixel intensities observed along a line of pixels incorporatingthe longest dimension of the suspect colony's image. The pixel positionsthat defined a change in intensity relative to the local background wereused to mark the margins of the colony image and to measure the colonydiameter (diameter distance was reported as the number of pixels locatedbetween the pixel points marking the colony margins).

The diameter of a gas bubble was determined using the second cultureplate image (100% back-side illumination). The imaging program analyzedfor changes in pixel color intensity observed along a line of pixelsincorporating the longest dimension of the bubble image. RGB(red/green/blue) image processing techniques were used, with the greenchannel providing the greatest contrast to identify gas bubbles in theparticular growth medium used in this culture device. The pixelpositions where a sharp decrease in pixel color intensity relative tothe local background color intensity occurred were identified and markedas the positions defining the dark annulus at the perimeter of thebubble. The diameter of the bubble was measured by counting the numberof pixels between the two identified pixel positions.

In the next step, the imaging program compared both the size andproximity of the gas bubble image (obtained from the second cultureplate image) to the size and position of the nearest colony image(obtained from the first culture plate image) and the bubble size andproximity criteria described above were applied. According to thecriteria, it can be concluded that gas bubbles 81, 82, and 83 in FIG. 6are all associated with colony 71 and, in addition, at least gas bubble81 is associated with colony 73. It can be concluded further that thereare no biogenic gas bubbles associated with colony 72 in FIG. 6.

In any case, various modifications may be made without departing fromthe spirit and scope of the invention. For example, one or more of therules described herein may be used with or without other rules andvarious subsets of the rules may be applied in any order, depending onthe desired implementation. These and other embodiments are within thescope of the following claims.

1. A method, comprising: using an imaging device to produce a firstimage of a thin film culture device, the culture device having a frontside having a transparent film cover sheet and a back side having atranslucent substrate; wherein the first image is produced whileproviding illumination to the front side of the device; wherein theculture device comprises an indicator compound that is converted by amicroorganism to a first product that is observable by a first color;wherein the culture device comprises a nutrient that can be converted toby a first type of microorganism to a gas; using the imaging device toproduce a second image of the thin film culture device, wherein thesecond image is produced while providing illumination to the back sideof the device; analyzing the first image to identify a microorganismcolony at a first location in the culture device; analyzing the secondimage to identify a first gas bubble at a second location in the culturedevice; and determining whether the first location is within apredetermined distance from the second location.